Human neutrophils use divalent cations for the stimulus-response coupling leading to the respiratory burst. We have studied the relevance of the extracellular versus intracellular calcium stores necessary for superoxide generation in neutrophils and neutrophil cytoplasts stimulated by N-formyl-methionyl-leucyl-phenylalanine (FMLP) and phorbol ester and our findings are as follows. (1) FMLP triggered superoxide anion (O2-) generation only in the presence of extracellular calcium, but phorbol myristate acetate (PMA) activation was independent of extracellular calcium. (2) inhibition of calcium transport channels with verapamil produced a dose-related inhibition of FMLP- and PMA-stimulated O2- production. Verapamil inhibition of FMLP-stimulated but not PMA-stimulated O2- was reversed simply by increasing extracellular calcium concentration. Cytosolic calcium loading with substimulatory concentrations of A23187 (0.3 to 0.6 mumol/L) (only in calcium-replete medium) could reverse the verapamil inhibition of PMA-triggered O2- generation. An inhibitor of intracellular calcium availability, 3,4,5-trimethoxybenzoic acid (TMB-8), similarly inhibited FMLP-stimulated and PMA-stimulated O2- production; this inhibition was reversible with A23187 calcium loading. (3) Verapamil, although it inhibited the functional oxidant response, had no inhibitory effect on fura 2-monitored cytosolic calcium rise induced by FMLP stimulation. (4) No cytosolic calcium increment was observed with PMA. O2- generation from enucleated polymorphonuclear leukocyte (PMN) cytoplasts was sensitive to inhibition by verapamil but was less so than O2- generation from intact PMNs. Cytoplasts, nearly depleted of cytoplasmic organelles and cytoskeleton, the potential sites of intracellular calcium stores, were markedly insensitive to the inhibitory effects of TMB-8.(ABSTRACT TRUNCATED AT 250 WORDS)